Abstract: :
Purpose: To determine the minimum functional region of the transactivationdomain for the rod photoreceptor specific bZIP protein NRL,in order to delineate biochemical mechanisms of transcriptionalactivation.Methods: A series of progressive N–terminal NRL deletionswere generated and cloned into the yeast two hybrid vector pHybLexZeo(Invitrogen). Six large and six small internal fragments ofNRL, and one internal deletion of NRL were also generated. Theseconstructs were used to transform yeast strain L40, which weresubsequently grown under selective conditions (minus–His,+zeo, +50mM 3–AT, +X–gal). Several of the NRL fragmentswere transferred into the pGBKT7 vector to verify their abilityto activate a reporter gene in a different strain of yeast.Interaction of NRL regions with TBP was examined using in vivoand in vitro co–immunoprecipitation assays.Results: Experiments using NRL deletion constructs in yeastdemonstrated that NRL transactivation was detected even whenamino acids 1 to 30 were removed. Subsequent deletions afteramino acid 75 showed little or no transactivation ability whengrown under selection on X–gal containing media. The smallestinternal NRL fragment containing transactivation function comprisedof amino acids 40–74. NRL amino acids 1 to 57 were unableto transactivate in the yeast assay. Full length NRL and TBPwere present in the same complex in bovine retinal nuclear extracts.The N–terminal region of NRL was observed to interactwith the C–terminal region of TBP.Conclusions: The minimal transactivation domain of NRL is fromamino acids 40 to 74 and a TBP binding domain overlaps withthis region. Our data suggests that the function of NRLâ|*128*|TMstransactivation domain is to recruit the general transcriptionmachinery to the promoter. These studies should provide a betterunderstanding of NRLâ|*128*|TMs function on the regulationof photoreceptor specific genes.